1. Field of the Invention
This invention relates to the field of molecule detection. More particularly, this invention relates to a system and method for using an active pixel sensor to optically detect the binding of probe molecules to target molecules.
2. Description of Related Art
In many applications, it is desirable to detect the presence of one or more types of target molecules in a sample. For samples of biological interest, such target molecules may include antigens, antibodies, or nucleic acids. One method for detecting target molecules in a sample involves the use of probe molecules that bind to only specific types of target molecules that may be present in the sample to form bound complexes. For example, if antigens are to be detected, antibodies specific for those antigens can be used as the probe molecules. Similarly, antigens can serve as the probe molecules to detect the presence of antibodies to that antigen. Additionally, nucleic acids can be detected by probe molecules having a sequence of nucleotides that is complementary to at least a portion of the nucleotide sequence in the nucleic acid, so that the probe molecules will hybridize with the target nucleic acids.
In order to detect target molecules, the probe molecules are disposed in a test site, and the sample is added to the test site to allow the probe molecules to bind with any target molecules present in the sample. The binding of probe molecules with target molecules can then be detected in a number of different ways, such as by measuring changes in the electrical or optical characteristics of the test sites.
Optical detection is potentially a very useful technique and can be accomplished in various ways. One approach is to apply electromagnetic radiation at a given wavelength, typically in the infrared, visible, or ultra-violet spectra, at which the absorbance of the bound complexes is substantially different than that of the unbound probe molecules. This approach, however, has the disadvantage of potentially requiring different wavelengths to be used depending on the probe molecules used and the target molecules to be detected. This approach can also suffer from low sensitivity.
A potentially better approach is to label the target molecules in a sample with fluorescent dyes (described herein as xe2x80x9cfluorophoresxe2x80x9d) before the target molecules are exposed to the probe molecules. Such fluorophores emit fluorescence radiation in a characteristic range of wavelengths when exposed to electromagnetic radiation at a characteristic excitation wavelength. Thus, the probe molecules will bind with target molecules to form bound complexes that are fluorescent. Binding can then be detected by illuminating the test sites with electromagnetic radiation at the excitation wavelength and sensing whether the bound complexes in the test sites emit fluorescence radiation.
Alternatively, instead of labeling the target molecules in the sample, the bound complexes can be exposed to a selective dye after they are formed. Selective dyes are dyes that exhibit substantial changes in their optical properties in the presence of bound complexes. For example, ethidium bromide is a fluorescent dye that exhibits an approximate 20-fold increase in fluorescence when intercalated into hybridized nucleic acids, as compared to when the dyes is unbound or bound only to unhybridized nucleic acids. In this way, the binding of molecular probes can be detected as either a substantial increase in fluorescence or a substantial increase in absorbance at the excitation wavelength.
In many cases, it is known that target molecules are present in a sample, but the nature of the target molecules is unknown. The probe molecules can then serve not only to detect the presence of target molecules in a sample but also to determine what the target molecules are. For example, a sample containing nucleic acids having an unknown nucleotide sequence may be exposed to multiple sets of probe molecules, with each set having a different nucleotide sequence. In this way, the nucleotide sequence of the nucleic acids can be determined from detecting which probe molecules bind to it.
Hollis et al., U.S. Pat. No. 5,653,939 disclose an apparatus that uses a plurality of different types of probe molecules disposed in an array of test sites to identify target molecules in a sample. To allow for optical detection of binding, the apparatus includes a charge-coupled device (CCD), in which each pixel is aligned with a respective test site. In this way, a change in the optical characteristics of the probe molecules in a given test site, due to binding with target molecules, can detected by the corresponding pixel. The array of test sites can be fabricated on the same chip as the CCD, or it can be provided in a separate glass plate. Optical detection of binding can occur in any of the ways described above, namely: (1) increased light attenuation caused by probe molecules binding with target molecules; (2) increased light attenuation at the excitation wavelength or increased fluorescence caused by ethidium bromide selectively intercalating into hybridized DNA; or (3) detection of fluorescence radiation after the probe molecules have bound to fluorescently-labeled target molecules. To better detect fluorescence radiation, each CCD pixel may be provided with a filter designed to block the excitation radiation and pass the fluorescence radiation.
Although the apparatus of Hollis et al. may potentially allow for the rapid detection and characterization of target molecules using a plurality of probe molecules, many shortcomings remain. Chief among these is inadequate portability. In particular, it is desirable in many applications to have a molecular detection apparatus that is portable, so that it can be brought to the location where the samples are collected.
However, CCDs, which are used in the apparatus of Hollis et al., have a number of characteristics that make them difficult to use in a portable molecular detection apparatus. First, CCDs are typically fabricated using a specialized process that is incompatible with conventional complementary metal oxide semiconductor (CMOS) processing. As a result, it is impractical to integrate the electronics necessary for amplification and image signal processing (which are best fabricated in CMOS) on the same chip as the CCD. Accordingly, when CCDs are used, portability is hampered by the need to include extensive off-chip signal processing electronics. Second, portability is made difficult in that CCDs require relatively complicated power supplies. Specifically, CCDs typically require voltages at three different levels (such as +15V, xe2x88x9215V, and xe2x88x928V) to operate. Third, CCDs and their associated electronics have a relatively high power dissipation. Fourth, CCDs are often cooled, such as by using fans, liquid nitrogen, or thermoelectrically. Accordingly, portability is further hindered by the need to provide additional cooling components that take up additional space, add weight, and increase costs. Moreover, the cooling components themselves require a substantial amount of power to operate, and they typically have their own power supplies, thereby further increasing the size and weight of the apparatus needed when using CCDs. CCDs are cooled for a number of different reasons. An important reason for cooling CCDs is to reduce noise, which is temperature dependent. Low noise operation allows CCDs to detect very weak signals. Notably, the ability to detect very weak signals is essential for molecule detection in biological samples, because only 1 femtomole or less of target molecules may be bound in a given test site. Thus, cooling is often needed in applications, such as molecule detection, where the CCD must be able to detect very weak signals. Additionally, because of the power dissipation in the CCD and associated electronics, cooling is often necessary to prevent damage to the components. Fifth, CCDs have another important limitation in that they read each pixel by transferring the photo-generated charge pixel by pixel until it reaches an amplifier at the edge of the device. Thus, a high charge transfer efficiency (CTE) is essential for good sensitivity in CCDs. Because the CTE decreases with increasing temperature, cooling is often required for good sensitivity. Sixth, to control exposure times of CCDs, external mechanical shutters are used. Such mechanical shutters take up space and require power to operate, thereby further hampering portability. Thus, it is difficult to make a CCD-based molecule detection apparatus highly portable because of the need to provide extensive electronics, complicated power supplies, cooling components, and an external mechanical shutter that take up a substantial amount space and require a substantial amount of power to operate.
In addition to poor portability, CCDs suffer from a phenomenon called xe2x80x9cblooming.xe2x80x9d Blooming occurs when a very strong signal saturates one pixel and causes charge to leak into neighboring pixels, thereby resulting in anomalous readings in the neighboring pixels. This can be a major problem when an array of test sites is used, because one test site may produce a very strong signal (e.g., because of probe molecules binding with fluorescently-labeled target molecules), whereas neighboring test sites may not have any binding and, thus, produce little or no signal. Accordingly, blooming from the strong signal may result in false positive readings in the neighboring pixels.
Finally, CCDs can also be substantially expensive because of the specialized techniques used to fabricate them.
Accordingly, a need exists to provide a molecular detection apparatus that allows rapid detection with high sensitivity and that is also portable.
In a first principal aspect, the present invention provides a system for testing a sample for the presence of target molecules. The system comprises a test site for receiving the sample, a source system for providing incident electromagnetic radiation, including radiation at a first wavelength, which radiation is directed to the test site, and an active pixel sensor. The active pixel sensor has a plurality of pixels, at least one of which is disposed for detecting electromagnetic radiation from the test site.
In a second principal aspect, the present invention provides a method for testing a sample for the presence of target molecules. The sample is added to a test site containing probe molecules that are able to bind to the target molecules to form bound complexes. The test site is illuminated with electromagnetic radiation, including radiation at a first wavelength. The electromagnetic radiation from the test site is detected with at least one pixel of an active pixel sensor.
In a third principal aspect, the present invention provides a method for identifying an unknown nucleotide in a nucleic acid, using a system comprising an active pixel sensor. The nucleic acid is added to a test site containing probe molecules that are able to hybridize with a nucleotide sequence adjacent to the unknown nucleotide in the nucleic acid. First, second, third, and fourth reporter molecules are added to the test site. The four types of reporter molecules comprise adenine, cytosine, guanine, and thymine conjugated to first, second, third, and fourth fluorophores, respectively. Each of the four fluorphores emits fluorescence radiation at a certain wavelength when exposed to electromagnetic radiation at a certain excitation wavelength. The test site is illuminated with electromagnetic radiation at each of the four excitation wavelengths in succession, and, during each such illumination period, a successive pixel of the active pixel sensor detects electromagnetic radiation from the test site.
In a fourth principal aspect, the present invention provides an improvement to a system for optically detecting the presence of specified molecules at specified locations. The improvement comprises an active pixel having a plurality of pixels. At least one of the pixels is disposed for detecting electromagnetic radiation from one of the specified locations.
In a fifth principal aspect, the present invention provides a method for monitoring the population of specified molecules at a location as a function of time. During a first time interval, at least one pixel of an active pixel sensor detects electromagnetic radiation from the location to obtain a first measurement. During a second time interval, the pixel detects electromagnetic radiation from the location to obtain a second measurement. The first and second measurements are then compared.